Lecture 2 - Examples of PMS

Question 1

What is the ratio of parental products in GC in an octad?

Answer 1

6:2

Question 2

What are the two ratios in PMS?

Answer 2

5:3 1 pair not same
4:4 2 pairs not the same
(secreted colonies in yeast tetrads)

Question 3

How do 4:4 aberrant ratios form?

Answer 3

through PMS
heteroduplexes formed in meiosis not repaired
visibilisation only visable after mitotsis

Question 4

How to 3:5 ratios form?

Answer 4

PMS

repair to one heteroduplex, mismatch repaired

Question 5

What are the four experiments by Szostak that show that original HJ required revision, because of DSBrepair?

Answer 5

1) dsbreaks dramatically increase recombination frequency
2) large gaps allow efficient integration
3) gap repair by recombination with intact homologous sequence
4) deletion to circular plasmid not repaired

Question 6

How was it shown experimentally that dsBreaks dramatically increase recombination frequency? (szostak)

Answer 6

1) circular plasmid inserted into yeast chromosome get few recombinants
2) when linearized plasmid inserted get many more recombinants

Question 7

How was it shown that large region in gaps of homology still allowed efficient integration?

Answer 7

1) showed 40 bp region of homology allowed integration of linearised plasmid

Question 8

How was it shown that gaps could be repaired by recombination with homolgous sequencE?

Answer 8

1) insertion of linearised plasmid with 40bp homologous regions and autonomously replicating sequence
Two results:
1) led to crossover and integration with chromosome
2) no crossover gap repaired from genetic information on the chromosome

Question 9

How was shown that deletion in circular plasmid not repaired?

Answer 9

circular yeast plasmid with region of homolgy with deletion

2)when integrated into the host chromosome regions of homolgy deletion not repaired

Question 10

What gene does dsb repair require and what happens in an individual defectivE?

Answer 10

RAD52

individuals defective in meiotic recombination

Question 11

What is the BSCR model of homolgous recombination?

Answer 11

1) recombination is initated by dsbreaks

2) involves double holliday junction

Question 12

What is the process dhj formed?

Answer 12

1) Dsbs are created by Spo11 (only in meiosis)
2) Resection by 5’ to 3’ exonuclease + helicases producing 3’ overhangs
3) binding of Rad51 and Dmc1 leads to strand invasion
4) Synthesis through break and branch migration
5) second end capture and synthesis
6) ligation leading to dHJ

Question 13

By what enzyme are JH cut by?

Answer 13

HJ resolvases to CO nCO products

Question 14

In resolution of HJ how are CO products formed?

Answer 14

draw

Question 15

In resolution of HJ how are nonCO products formed?

Answer 15

draw

Question 16

If heteroduplexes repaired before dHJ formed how effect gene conversion?

Answer 16

2:6

Question 17

In heteroduplex repair where is it most liely to occur?

Answer 17

on the chromatid where DSB occured because mismatch repair works on a strand containg a free end

Question 18

What are the main types of enzymes involved in crossing over?

Answer 18

endonuclease to make dsb, 5’ to 3’ exnuclease, helicases, recombinase, polymerase, ligase, resolvase

Question 19

Why bother with gene conversion?

Answer 19

Meiosis = generates genetic variation e.g. results gene conversion  without  crossing over can only be replicated by double cross over rare if loci are closely linked
Mitosis = loss of heterozygosity and disease, cancer

Question 20

What are the features of Spo11?

Answer 20

• conserved
• meiosis specific
• low sequence specifity

Question 21

Why Spo11 not in mitotic cells?

Answer 21

DSB and dhj lead to LOH cancer/disease

Question 22

What recombinase catalyses strand exchange in vitro?

Answer 22

RecA

Question 23

What was Sehorn experiment to show strand exchange in vitro?

Answer 23

Used purified human-meiosis specific recombinase Dmc1

1) added Dmc1 to css and lds
2) took samples at different time points
- css and lds
- jm
- nc and lss
3) analysed products by gel electrophoresis

Question 24

What challenged of dHJ?

Answer 24

finding correct sequences

why use homologous rather than sister chromatids

Question 25

How is the correct homolgous sequence found?

Answer 25

recombinases allow homolgy search within 3stranded structure

Question 26

Why do think that NCO products mostly generated via a hj independednt mechanism and what is this mechanism>

Answer 26

• most NCO products are detected before HJ formed

- synthesis dependent strand annealing

Question 27

What are the key points of SDSA?

Answer 27

1) newly synthesised DNA displaced from template anneals to complimentary broken end
2) donor chromatid remains unchangd

Question 28

What is strand return facilitated by and what proteins?

Answer 28

facilitated by bubble migration

helicases stop invading strand from synthesising from template DNA

Question 29

What is more frequent during meiosis, gene conversions or co?

Answer 29

gene conversions

Question 30

What are the outcomes of DSB in meiosis?

Answer 30

1) repaired using homolgous chromosomes
2) leading to few dHJ -> CO
3) or SDSA -> non cross overs

Question 31

What are the outcomes of DSB in mitosis?

Answer 31

1) repair using sister chromatid
2) leads to very few dHJ -> CO and nCO
3) OR SDSA leads to nonCO

Question 32

How can view CO and GC in meiotic cells?

Answer 32

immunoflourence analyss of human pachytene spermatocyte
antibodies stain
-synaptonemal complexes - red
-centromere - blue
-CO - green
show all bivalents have at least 1 CO nomore 3

Question 33

How is the frequency of crossing over & dsb regulated?

Answer 33

by spo11
where Co occur, not at Spo11
where DSB form, not Spo11 nearby sites